Enhancing catalytic performance of porcine pancreatic lipase by covalent modification using functional ionic liquids

Article abstract of DOI:10.1021/cs400404f

Various functional ionic liquids (ILs) composed of different cations and anions were activated with carbonyldiimidazole and then covalently linked onto porcine pancreatic lipase (PPL) through lysine coupling. Catalytic performances, such as activity, thermostability, and enantioselectivity were improved successfully, as was investigated in p-nitrophenyl palmitate (pNPP) hydrolysis and racemic 1-phenethyl acetate hydrolysis reaction. The correlation between catalytic performance and modification of IL was studied by catalytic and spectroscopic data, which showed improvement of catalytic performances to a different extent. Hydrolytic activity was enhanced by ILs with chaotropic cations and kosmotropic anions (e.g., more than 4-fold with [choline][H ₂PO₄]). Modifications by ILs bearing kosmotropic cations and chaotropic anions contribute to lipase thermostability and enantioselectivity (e.g., modification with [HOOCBMIm][Cl] showed a 12-fold thermostability increase at 60 C and more than 7-fold enantioselectivity enhancement than native enzyme). The Matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry experiments suggest that ILs bind with lipase protein. Conformation changes were confirmed by fluorescence spectroscopy, and circular dichroism spectroscopy.

Full text of DOI:10.1021/cs400404f

Research Article
pubs.acs.org/acscatalysis
Enhancing Catalytic Performance of Porcine Pancreatic Lipase by
Covalent Modification Using Functional Ionic Liquids
Ru Jia,† Yi Hu,† Luo Liu,‡ Ling Jiang,† Bin Zou,† and He Huang*,†
†State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering,
Nanjing University of Technology, Nanjing 210009, China
‡Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing
100029, China
S
* Supporting Information
ABSTRACT: Various functional ionic liquids (ILs) composed
of different cations and anions were activated with carbon-
yldiimidazole and then covalently linked onto porcine
pancreatic lipase (PPL) through lysine coupling. Catalytic
performances, such as activity, thermostability, and enantiose-
lectivity were improved successfully, as was investigated in p-
nitrophenyl palmitate (pNPP) hydrolysis and racemic 1-
phenethyl acetate hydrolysis reaction. The correlation between
catalytic performance and modification of IL was studied by
catalytic and spectroscopic data, which showed improvement
of catalytic performances to a different extent. Hydrolytic
activity was enhanced by ILs with chaotropic cations and
kosmotropic anions (e.g., more than 4-fold with [choline]-
[H2PO4]). Modifications by ILs bearing kosmotropic cations and chaotropic anions contribute to lipase thermostability and
enantioselectivity (e.g., modification with [HOOCBMIm][Cl] showed a 12-fold thermostability increase at 60 °C and more than
7-fold enantioselectivity enhancement than native enzyme). The Matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometry experiments suggest that ILs bind with lipase protein. Conformation changes were confirmed by fluorescence
spectroscopy, and circular dichroism spectroscopy.
KEYWORDS: enzyme catalysis, ionic liquids, protein modifications, specific ion effects, structure−activity relationship
of native biocatalysts,11,12 also enzyme properties may be
INTRODUCTION
■
improved via chemical modification compatible with a proper
Over the past decade, scientific and technological advances
have established biocatalysis as a practical and environmentally
friendly alternative to traditional metallo- and organocatalysis in
immobilization.13,14 Several modifiers that improve the catalytic
performance of PPL have been reported in recent years.
Improving 20% thermostability was observed by grafting Z-
chemical reactions, both in the laboratory and on an industrial
scale.1 Lipases are important biocatalysts that catalyze various
reactions, such as esterification, interesterification, aminolysis,
and hydrolysis, which produce numerous important chemicals
including alcohols, esters, acids, and amine.2,3 Lipases tend to
form bimolecular aggregates even at very low concentrations,
these aggregates have very different properties when compared
with the monomeric form of the lipases, this makes the study of
the enzyme very complex and difficult to reproduce or
understand the differences between different researches,4−7
and dimers that may be broken by detergents (e.g., Triton X-
100).8 Porcine pancreatic lipase (PPL) is one of the most
widely used lipases in biotransformation reactions because it is
cheaper and more accessible compared with other commercial
enzymes; however, low activity and stability of PPL are usually
observed.9
proline on PPL in the temperature range from 20 to 50 °C with
respect to native enzyme, with no significant increase in
hydrolysis activity.15 Onefold thermostability and 20%
hydrolysis activity at 50 °C of PPL increased when modified
with phthalic anhydride.16 However, simultaneous and
noticeable increase in both activity and stability were not
obtained by the aforementioned modifiers nor was enantiose-
lectivity assayed. Therefore, developing a novel and efficient
chemical modification method is necessary to further improve
the catalytic performance of PPL.
Ionic liquids (ILs), which act as solvents or additives, have
been widely used in enzymatic catalysis and present enzymes
with excellent activity and stability because of their environ-
mentally friendly and desirable features.17−19 In our previous
Chemical modification is an important approach to alter
protein function by introducing non-natural fragments into
proteins.10 This specific modification can expand the efficiency
Received: May 29, 2013
Revised: July 15, 2013
Published: July 17, 2013
© 2013 American Chemical Society
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Research Article
Scheme 1. Chemical Modification of PPL with Functional Ionic Liquids
studies, functional ionic liquids were synthesized and grafted
onto the surface of mesoporous SBA-15; high activity and
stability of immobilized PPL was obtained.20−23 Recently,
stored in solution at 4 °C or used directly after modification.
Enzyme concentration was determined via the BCA method
using bovine serum albumin (BSA) as standard.27 The
modification degree was investigated using the trinitrobenzene
sulfonate (TNBS) assay procedure.28
̀
Bastien Doumeche et al. reported that IL covalently bound to
formate dehydrogenase to improve its stability and activity in
ILs, and found a higher modified degree from a more
chaotropic cation, contributing to significantly improved
activity and stability.24,25
Enzyme Activity. The activity of the enzyme to catalyze the
hydrolysis of p-nitrophenyl palmitate (pNPP) to p-nitrophenol
(pNP) was assayed following the procedures described by
Kordel et al.,29 with slight modification. The substrate solution
was prepared by mixing one volume of a solution of pNPP
(16.5 mM) with 9 volumes of buffer (50 mM phosphate pH
7.5) containing 0.4% (w/v) Triton X-100 and 0.1% (w/v)
Arabic gum. Then a volume of 10 μL of an appropriate dilution
of the enzyme solution was added to 240 μL substrate solution
to start the reaction at 25 °C, the pNP formation was
monitored at 405 nm using a Model 680 Microplate Reader
(Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.). Under the
conditions used, the extinction coefficient of p-nitrophenol was
8620 M−1 cm−1. One unit of lipase activity was defined as the
amount of lipase releasing 1 μmol of p-nitrophenol from pNPP
per minute.
Enzyme Thermostability. The enzyme solution in
deionized water was incubated at 30, 40, 50, and 60 °C,
respectively. Samples of 30 μL were taken periodically for
activity assay at 25 °C, as previously mentioned. The time-
dependent loss in activity was used to calculate the half-life for
each enzyme solution.
Kinetic Measurements. The reaction was performed as
described in “Assay of Enzyme Activity”. The substrate
concentration in the reaction mixture varied between 0.08
and 0.83 mM at a constant enzyme concentration (0.1 mg/mL)
for 4 min. The maximum rate (Vmax) and the Michaelis
constant (KM) of the enzyme were determined by Hanes−
Woolf plots.
Enantioselectivity Measurements. (R,S)-1-phenethyl
acetate (60 mM) was added to 10 mL aqueous buffer (100
mM, pH 7.5), followed by ultrasonic dispersion for 10 min,
before the enzyme (5 mg) was finally added. The mixture was
stirred in 150 rpm at 25 °C. At an appropriate time interval,
aliquots (0.2 mL) were withdrawn, then both the 1-phenyl-
ethanol-produced and the residual ester were extracted into
ethyl acetate (0.2 mL). The organic layer was dried over
Na2SO4, and then subjected to HPLC analysis on a chiral
column (Chiralcel OZ-H, hexane/2-propanol = 98/2). In each
case, the nonenzymatic reaction was subtracted from the total
reaction.
In this study, PPL was first modified by a series of ILs
composed of different cations and anions (Scheme 1). The
modification degree, activity, kinetic parameters, thermal
stability, and enantioselectivity were investigated. This study
aims to develop a novel, efficient, and practical method to
enhance the catalytic performance of lipase and propose useful
guidelines for designing functional ILs applied to modify lipase.
EXPERIMENTAL SECTION
■
Unless otherwise noted, all reagents are of analytical grade or
higher. PPL (type II), p-nitrophenyl palmitate (98%), p-
nitrophenol (99.5%), and 2,4,6-trinitrobenzenesulfonic acid
solution (5% w/v in H2O) were purchased from Sigma-Aldrich
China Inc. (R,S)-1-Phenethyl acetate was prepared according to
standard procedure. Bisuccinic acid (BCA) protein assay
reagent kit was obtained from Pierce Chemical Co. (Rockford,
IL, U.S.A.). All seven functional ILs (99%, HPLC) were
purchased from Shanghai Cheng Jie Chemical Co., Ltd.
Carbonyldiimidazole (97%) was from Aladdin Chemistry Co.,
Ltd., matrix-assisted laser desorption/ionization-time-of-flight
(MALDI-TOF) mass spectra were obtained using a BRUKER
Auto FLEX-T2 apparatus.
Enzyme Preparation. The PPL solution was obtained by
solubilizing the enzyme powders (2 g) in 50 mL of the
deionized water, with magnetic stirring for 30 min at 4 °C.
Solubilization was followed by centrifugation and subsequent
concentration with ammonium sulfate precipitation. Finally,
PPL was transferred into a 10 kDa dialysis membrane to
remove excess salt. The purity of the enzyme was confirmed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
Chemical Modification. Two milliliters anhydrous dimeth-
yl sulfoxide (DMSO) containing IL (500 mM) and carbon-
yldiimidazole (500 mM) were stirring in dark for 2 h at 25 °C
to activate the functional IL.26 After that, 200 μL reactant
without separation were added into 10 mL of PPL solution
(100 μM) in deionized water (pH 7.0), with magnetic stirring
for 8 h at 4 °C. The modified PPL was purified by ultrafiltration
in deionized water using a 10 kDa membrane. Enzymes were
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Table 1. ILs Used in This Study
number
ionic liquid
cation
anion
IUPAC name
1a
1b
1c
1d
1e
1f
[HOOCMMIm][Cl]
[HOOCEMIm][Cl]
[HOOCBMIm][Cl]
[HOOCBMIm][H2PO4]
[HOOCMMIm][PF6]
[choline][NO3]
chaotropic
chaotropic
kosmotropic
kosmotropic
chaotropic
chaotropic
chaotropic
chaotropic
chaotropic
chaotropic
kosmotropic
chaotropic
chaotropic
kosmotropic
3-(2-carboxymethyl)-1-methylimidazolium chloride
3-(2-carboxymethyl)-1-ethylimidazolium chloride
3-(2-carboxymethyl)-1-butylimidazolium chloride
3-(2-carboxymethyl)-1-butylimidazolium dihydrophosphate
3-(2-carboxymethyl)-1-methylimidazolium hexafluorophosphate
choline nitrate
1g
[choline][H2PO4]
choline dihydrophosphate
Fluorescence Spectroscopy. The fluorescence spectra of
the dilute enzymes (0.5 μM) were monitored using a
spectrofluorometer (PerkinElmer LS55, U.S.A.) at 25 °C and
excited at 270 nm. The emission registered from 300 to 400 nm
using a 5-nm bandwidth in both excitation and emission paths.
Circular Dichroism (CD) Spectroscopy. Circular dichro-
ism (CD) (190−250 nm) spectra were recorded on a JASCO-
J810 Spectropolarimeter (Jasco Co., Japan) in a cell with 1 cm
light path length at 25 °C. The scanning rate was set at 50 nm/
min and the spectra were the average of three readings of a
highly dilute enzyme solution (0.5 μM). Baseline correction
was automatically carried out with the deionized water
spectrum during the complete collection time. The secondary
structure parameters of the enzymes were computed using the
Jwsse32 software according to the reference.30
Figure 1A and 1B (taking PPL-1a for example) show that the
modification degree was proportional to the molar ratio and
RESULTS AND DISCUSSION
■
Design of Different Cations and Anions of ILs Used in
This Study. Specific ion effects have been well-known in
biology for over a century.31 In recent years, numerous reports
focused on the relationship between the ionic nature of ILs and
the enzyme properties, with kosmotropic ions having high
surface charge density and strong hydration, whereas the
opposite conditions were observed in chaotropic ions.32−34 In
this study, seven kinds of ILs containing different typical
kosmotropic/chaotropic ions were designed, as listed in Table
1. The chaotropicity of cations is in the order of choline+>
MMIm+> EMIm+> BMIm+, with the first three being
chaotropes and the last one being kosmotropic cation.35 The
Figure 1. Modification degree and activity of PPL-1a (line + symbol,
activity; column, modification degree). Reaction conditions: (A)
different molar ratios of modifiers for 10 h and (B) different
modification time at optimized molar ratio.
chaotropicity of anions vary in the order of PF6 > NO3 > Cl−,
−
−
36
−
whereas H2PO4 is considered a kosmotrope.
Modification of PPL with ILs. PPL is a globular protein
composed of a single chain of 449 amino acids, with a
molecular weight of 50−52 kDa. The open conformation of the
lid domain in PPL is stabilized by hydrogen bonds between the
lid and colipase, which allows the access of the substrate to the
active site of the lipase. Colipase is a small protein cofactor with
molecular weight of 10 kDa which anchors lipase to the bile
salts to the lipid/water interface.9 Also, commercial preparation
of PPL contains several contaminant proteins with lipolytic
activity, among them are three main components: the actual
PPL, a lipase of 33 kDa and a protein with approximately 25
kDa, those may give very different results when they are used as
biocatalysts.37−39 So the purification of crude PPL is necessary
before any modification (Supporting Information, Figures S1).
Various functional ILs were activated with carbonyldiimida-
zole before being covalently linked onto the lysine residues.26
The modification degree was controlled by the amount (molar
ratio) and time of the activated ILs grafted onto lysine residue.
time of activating groups to amino groups, and plateau values
were observed at the ratio of 100:1 and 8 h. The enzyme with a
higher modification degree showed higher activity. The other
ILs modified on PPL showed the same tendency (Supporting
Information, Figures S2−S7). Therefore, considering both
modification degrees and activities, the molar ratios of 100:1
and 8 h of activating ILs to PPL were the appropriate
concentration and modification time, respectively.
MALDI-TOF mass spectra experiments were conducted to
confirm the IL binding with the lipase protein. The number of
modified lysine residues is determined in Figure 2. The mass
spectrum (MS) of the native enzyme presents a single peak at
m/z 51,725 (Figure 2A). The grafting of cation (IL-1a) onto
PPL resulted in a heterogeneous population of seven proteins
containing 7 to 13 modified lysine residues (Figure 2B). Each
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Anions also have an important function in modification. For
PPL-1a and PPL-1e, PPL-1c and PPL-1d, and PPL-1f and PPL-
1g, their modified ILs have the same cations. A higher
modification degree was obtained with a more kosmotropic
anion. A microaqueous phase surrounds the enzyme molecules,
and the modifier should penetrate this aqueous layer for direct
contact with the enzyme for modification. Thus, the activated
cation will be easy to graft onto the enzyme with a more
kosmotropic anion by preferential hydration.
Hydrolytic Activity Assay. Table 3 shows that the
activities of all seven modified enzymes compared with native
a
Table 3. Kinetic Parameters of Enzymes
enzyme
Vmax (U/g)
KM (mM)
kcat/KM (min−1 mM−1
)
PPL
15.43 1.56
53.52 2.34
47.89 2.57
22.17 2.11
28.29 2.03
39.98 3.03
43.51 2.36
64.18 2.89
0.297 0.006
0.345 0.007
0.315 0.005
0.274 0.005
0.286 0.006
0.321 0.005
0.332 0.007
0.349 0.008
2.69
8.02
7.86
4.19
5.12
6.44
6.78
9.51
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
Figure 2. Results of MALDI-TOF mass spectra experiments on PPL
(A) and PPL-1a (B).
a
Conditions: substrate concentrations varied between 0.08 mM and
0.83 mM, protein content (0.1 mg/mL), sodium phosphate buffer
(pH 7.5, 50 mM) were performed at 25 °C for 4 min.
cation (IL-1a) led to the increased mass of 123, and PPL
contains 21 lysine residues (PDB ID 1ETH), numbers or
degree of modification could be calculated with the data
showed above; the other MS were shown in Supporting
Information Figures S8−S10.
enzyme in aqueous solution varied broadly. PPL was activated
by the modification of all seven ILs. PPL was activated highest
at 415.94% (by [choline][H2PO4]) and lowest at 143.68% (by
[HOOCBMIm][Cl]) of the activity relative to native enzyme
in pNPP hydrolysis. In the presence of ILs with the same
anions, the enzyme activity decreased in the order of PPL-1a>
PPL-1b> PPL-1c, whereas the order was PPL-1a> PPL-1e,
PPL-1g> PPL-1f, PPL-1d> PPL-1c for ILs holding the same
cations.
Table 2 shows the average modification degree investigated
using TNBS assay procedure, which agree with the MS results.
Table 2. Degree of Amino Groups Modified on PPL
Molecule
numbers of amino groups
degree of amino groups modified
a
enzyme
modified (MS)
(%) (TNBS)
The results indicate that when owning the same anions,
modified ILs with more chaotropic cations produce higher
improvement of activity. This result is probably correlated with
the higher modification of lysine residues compared with
kosmotropic cation, which is suspected to prevent more
efficient lipase unfolding. The specific ion effect of anions on
lipase showed that the more kosmotropic the anion was, the
higher the activity observed.40 Similar results were obtained in
this study. Kosmotropic anion possibly facilitates more cations
to graft onto the enzyme surface with improved hydration,
resulting in higher modification and activity. The KM values
were slightly altered, and KM slightly increased after
modification, except for PPL-1c and 1d. The catalytic efficiency
(kcat/KM) of modified enzymes increased up to 1.56 to 3.54
than native enzyme because of the slight change in KM values. A
slight decrease in KM of PPL-1c and 1d were observed
compared with the other enzymes. These changes of lipase after
ionic liquids modification could be attributing to the open-
closed equilibrium of the lipase toward the active form.
Thermostability Assay. The thermostabilities of both
native enzyme and modified enzymes were studied by
measuring the residual activities of the enzymes after incubation
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
7−13
5−10
3−6
53.4 2.8
36.8 2.4
22.6 1.2
27.9 2.1
41.2 2.3
46.3 2.6
65.3 3.5
3−7
6−11
7−12
9−16
a
Conditions: 0.25 mL of 0.01% TNBS was added to 0.75 mL
phosphate buffer (0.025 mol/L, pH 8.2, containing 66 μg/mL
enzymes) then incubation at 37 °C. After 2 h, 0.5 mL sodium dodecyl
sulfate solution (10%) and 0.25 mL hydrochloric acid solution (1 mol/
L) were added then measured at 335 nm in a UV-1200.
For the same anion, the more chaotropic cations resulted in
higher modification degrees. These values were obvious for
PPL-1a, PPL-1b, and PPL-1c, whose modification degrees were
53.4%, 36.8%, and 22.6%, respectively. Doumec
̀
he24,25 reported
that this higher number of grafted residues is partially explained
by lower steric hindrances induced by the cation size and the
alkyl chain flexibility.
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Research Article
in aqueous solution for appropriate periods at different
temperatures (30, 40, 50, and 60 °C). Table 4 shows that
Table 5. Results of Lipase-Catalyzed Hydrolysis of (R,S)-1-
Phenethyl Acetate
a
Table 4. Thermostability of Modified PPL Compared with
Native Enzyme in Aqueous Solution
half life (min)
enzyme
30 °C
40 °C
50 °C
60 °C
0.5
b
b
enzyme
time (h)
ees (%)
eep (%)
conv. (%)
E value
PPL
75
75
3
27
50
62
73
60
93
52
41
2
10
31
32
50
35
62
31
22
1
2
10
13
24
20
31
12
10
PPL
5
3
4
5
5
4
4
3
75
88
85
80
82
85
86
89
89
92
93
98
98
97
95
94
46
49
48
45
46
47
48
49
39
71
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
4
7
6
5
3
3
3
2
5
4
3
2
3
3
3
4
2
2
1
2
3
1
3
2
1
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
85
76
100
85
>200
>200
183
113
101
130 10
105
66
8
4
a
Conditions: (R,S)-1-phenethyl acetate (60 mmol), enzymes (5 mg),
modified enzymes exhibited higher stability. The anions and
cations significantly influenced the thermostabilities, rather than
the modification degree, compared with the effect on hydrolytic
activity. At each incubation temperature, the lipase stabilized in
the presence of imidazolium ILs with the same anion varied in
the order of: PPL-1c> PPL-1b> PPL-1a. For ILs with the same
cations grafted on PPL, improved thermostability was observed
with a more chaotropic anion.
and sodium phosphate buffer (10 mL, pH 7.5, 100 mM) were
performed at 25 °C and 150 rpm. Calculated by eep and ees. E = ln[1
b
− c(1 + eep)]/ln[1 − c(1 − eep)], here c means conv., which was
calculated by the following formula: c = ees/(ees + eep).44,45
kosmotropic cation leads to higher enantioselectivity and the
hydrophobicity of modified ILs increase with increasing length
of the alkyl group attached on the cation. When grafting onto
enzyme, cations have a highly important function in affecting
enantioselective performance. Higher enantioselective effi-
ciency was obtained with a more kosmotropic cation.
Kosmotropic ion interacts more strongly with water, thus,
this ion can help maintain the enzyme conformation and
stabilized the enzyme.49,50 Also, when grating more hydro-
phobic cations, they can make more contribution to the lid
opening and active conformation forming,51,52 those may be
the reason that PPL-1c and PPL-1d own better enantiose-
lectivity than the other modified enzymes. It was found that the
enantioselectivity was enhanced by the ILs which acted as
additives or solvents containing kosmotropic anions,53,54 our
experiments showed opposite results that chaotropic anions
help increase enantioselectivity. The reason may be that ILs as
modifiers and cations grating on enzyme are more crucial in
affecting the enantioselectivity of the enzyme. PPL-1e also
showed great enantioselectivity, the possible explanation is that
When ILs were grafted covalently onto the enzyme surface,
bulk water was perturbed by the cations. BMIm+ is a
kosmotropic ion with a high charge density that interacts
more strongly with water than MMIm+ and EMIm+. Thus,
BMIm+ tends to protect essential water molecules, which
stabilizes lipase. Anion has a large effect on enzyme stability.41
The results emphasized that better thermostability can be
observed, such as PPL-1a and PPL-1e, by changing only the
anion to a more chaotropic one. Although MMIm+ is a
−
chaotropic cation, with a more chaotropic PF6 , this cation
showed excellent thermostability, as previously found for
lipases.42,43
Enantioselectivity Assay. All the eight enzymes have been
tested in the hydrolysis of racemic 1-phenethyl acetate. The
data in Table 5 show that using grafted ILs on PPL in the
lipase-mediated resolution of racemic 1-phenethyl acetate can
significantly enhance activity and enantioselectivity. The PPL-
1a and PPL-1g showed high hydrolytic activity, achieving
higher conversion with almost half time than native enzyme, in
contrast, the PPL-1c and PPL-1d displayed a very low activity
similar to native enzyme, and the results were in agreement
with the consequence of hydrolysis of pNPP. From the data
summarized in Table 5, seven different modified enzymes
increased enantioselectivity to a different extent, PPL-1a and
PPL-1g owned great hydrolytic activity with only slight increase
of enantioselectivity, while PPL-1c which owned more than 7-
fold enantioselectivity compared with that of native enzyme,
furnishing (R)-1-phenylethanol in 98.3% eep, corresponding to
an E value of 290, even though the hydrolytic activity without
significant improvement.
All tested hydrolysis reactions of racemic 1-phenethyl acetate
employing the seven different modified enzymes significantly
increased enantioselectivity than catalyzing with native enzyme.
The results of increased enantioselectivity may be the
introduction of the ILs chemical modification on enzyme
caused a conformational change on the enzyme and form the
active open-lid conformation of the enzyme.46−48 In the case of
PPL-1a, PPL-1b, and PPL-1c, with the same anion, more
−
modified IL contain PF6 , which is typically stabilizing enzyme
and help achieve high enantioselectivity.55
Intrinsic Fluorescence Analysis. Investigation on any
possible conformational changes induced by IL modification
may shed some light on the effect of ILs on enzyme
performance. Fluorescence spectroscopy is used to follow the
changes in the maximal intensity of fluorescence (Imax) and the
maximal emission wavelength (λmax).56 Figure 3A shows the
fluorescence spectra of PPL and three differently modified
enzymes in water. Figure 3B shows the spectra of enzymes
(mentioned in Figure 3A) which incubated at 40 °C for 30 min.
The maximum intensity for all the spectra has been normalized
with respect to the initial spectrum obtained from PPL in water
at 25 °C.
PPL exhibited an initial λmax of 350 nm in water. After
modification, it is noticeable how the fluorescence spectra of
modified enzymes were clearly modified compared with the
spectra of native enzyme. ILs with the same anion grafting on
enzyme exhibited blue shift in λmax and an increase in Imax
(Figure 3A, Supporting Information Figure S11A). Chaotropic
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Figure 3. Fluorescence spectra: (A) three imidazolium ILs with Cl− as
the anion and (B) enzymes incubated at 40 °C for 30 min compared
with (A).
Figure 4. Far-UV CD spectra of PPL and modified PPL in water: (A)
three imidazolium ILs with Cl- as the anion and (B) enzymes
incubated at 40 °C for 30 min compared with (A).
maintain native conformation to a great extent, while MMIm+ is
chaotropic cation.60
cation grafting on enzyme resulted in a higher maximal
intensity than kosmotropic ones, which could be the amount of
modification degree, the results showed that protein changes to
an active conformation with high activity.57 After incubation at
40 °C for 30 min, the fluorescence spectra of native enzyme
clearly shifted from the initial spectra, while spectra of modified
enzymes had negligible changes (Figure 3B, Supporting
Information Figure S11B). The decreased Imax and the red
shift of λmax observed in PPL spectra correspond to a classical
unfolding process of proteins which were accompanied by an
activity loss,58 while little changes of spectra of modified
enzymes showed the ability to stabilize enzyme (Table 4).
Circular Dichroism Analysis. Circular dichroism spectra is
one of the most widely used techniques for determining the
structure of proteins, making it possible to quantify conforma-
tional modifications in the 3D structure from changes in the
CD spectra. In particular, the line shape analysis of far-UV CD
spectra (190−250 nm wavelengths) may allow identifying
modification-induced structural changes of α-helice, β-sheet, β-
turn and random coil in proteins, respectively.59 The far-UV
CD spectra of the enzymes indicated that the protein has a
particular distribution of the secondary structure in each
condition. ILs with same anion grafted on PPL caused variation
of protein conformation (Figure 4A, Supporting Information
Figure S12A). After incubation at 40 °C for 30 min, the
noticeable conformation shift of PPL occurred, as shown in
Figure 4B. The conformations of modified enzymes changed
differently, especially the spectra of PPL-1a. Compared with the
other two, PPL-1c slightly shifted. This result can be attributed
to the kosmotropicity of BMIm+, which helps preserve critical
water molecules in the microenvironment of the enzyme and
The secondary structures from the CD spectra were
computed using the Jwsse32 software following the method
of CD-Yang.Jwr (Table 6). The secondary structure distribu-
tion of native enzyme was determined from an aqueous
solution at 25 °C, which indicates that the α-helix content
agrees with the reported data.61 All the modified enzymes lower
Table 6. Secondary Structure Percentages of Enzymes from
CD Spectra: 25 °C without Incubation (A) and Incubation at
40 °C for 30 min (B)
α-helix (%)
β-sheet (%)
β-turn (%)
random (%)
(A)
28.0
31.4
32.3
38.8
34.8
33.3
30.2
34.1
(B)
PPL
27.4
20.2
18.2
17.5
18.1
19.6
21.4
19.5
17.1
17.3
18.8
14.4
16.9
16.8
17.5
15.2
27.5
31.1
30.7
29.3
30.2
30.3
30.9
31.2
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
PPL
14.6
12.7
12.1
16.2
11.8
12.3
11.4
12.8
20.1
33.9
36.5
39.8
37.6
33.9
33.5
35.5
23.1
11.5
14.6
13.8
13.2
18.2
14.6
13.4
42.2
41.9
36.8
30.2
37.4
35.6
40.5
38.3
PPL-1a
PPL-1b
PPL-1c
PPL-1d
PPL-1e
PPL-1f
PPL-1g
1981
dx.doi.org/10.1021/cs400404f | ACS Catal. 2013, 3, 1976−1983

ACS Catalysis
Research Article
α-helix content but higher β-sheet content in comparison with
native enzyme, especially PPL-1c. The analysis of the CD
spectrum of the PPL incubated at 40 °C for 30 min (Figure 4B,
Supporting Information Figure S12B) showed a decline in both
α-helix and β-sheet secondary structures, which agree with an
unfolded protein state. This loss of native enzyme con-
formation is clearly correlated with poor thermostability, as
shown in Table 4. Enzyme modification preserves catalytic
activity, which is reflected by the maintenance of the secondary
structure of PPL. After modification, the structure did not
change significantly. In the same way, the loss of α-helix
content accompanies a significant increase in the β-sheet
content of modified enzymes with time. These results show
how the active enzyme conformation was protected by IL
grafting against deactivation, displaying high stability and
enantioselectivity as seen from a comparison with Tables 4
and 5.
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ACKNOWLEDGMENTS
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This research was supported by the National Science
Foundation for Distinguished Young Scholars of China
(Grant No. 21225626), National Natural Science Foundation
of China for Young Scholars (Grant No. 20906049), Key
Project of National Natural Science Foundation of China
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